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Figure A1. Comparison of the concentration-response curves for P2X4Rs or NMDARs expressed individually (depicted with solid lines) or together (depicted with dotted lines) in Xenopus leavis oocytes. (a) ATP concentration-response curves. EC50 values obtained from ATP-concentration curves of individual P2X4 and P2X4 coexpressed with NMDARs were not significantly different. (b–d) Glutamate-concentration response curves. EC50 values were not statistically significantly different for Glu-concentration response curves for individual GluN2A, GluN2B, GluN2C (solid lines) and each NMDAR subtype coexpressed with P2X4 (dotted lines). P2X4 and NMDARs were injected at respectively 20 ng and 10 ng cRNAs. Data represent Mean ± SEM. Statistical analysis performed using Exact sum-of-squares F-test. (a) p > 0.5; n = 9–12; (b) p > 0.5; n = 9–12; (c) p > 0.3; n = 9–12; (d) p > 0.5; n = 9–12.
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Figure A2. Application of ATP agonist does not affect Glu responses when NMDARs are expressed alone and conversely, application of Glu does not modulate ATP responses when P2X are expressed alone. (a) Representative current recorded from an individual oocyte GluN2B-containing NMDARs responding to 100 µM: Glu (left), ATP (middle), or Glu + ATP (right) are shown.; (b) Bar graphs representing the current obtained from application of agonists, normalized to the Glu response for each GluN2B-expressing oocyte.; (c) Representative current recorded from an individual oocyte expressing only P2X2 responding to 100 µM: ATP (left), Glu (middle), or Glu + ATP (right) are shown. (d) Bar graphs representing the current obtained from application of agonists, normalized to the ATP response for each P2X2-expressing oocyte. The data are expressed as mean ± SEM. ns > 0.05 (one-way ANOVA).
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Figure A3. P2X4-mediated NMDAR inhibition depends on the P2X4 CT domain. Bar graphs representing the mean of the amplitude of NMDARs responses in CfRS before and after P2X4-377TR activation by ATP. All values were normalized to the Glu response obtained before P2X4-377TR stimulation. None of the Glu responses after ATP application were statistically significantly different (p > 0.05) from the baseline response (before ATP application) for any GluN2 subunit. The data are expressed as mean ± SEM. Statistical analysis performed using a one-way ANOVA test.
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Figure A4. P2X2–NMDAR inhibition is voltage-independent. (A) Representative currents recorded at different holding potentials (VH) from an individual oocyte coexpressing NMDARs and P2X2 responding to 100 µM: Glu (left), ATP (middle), or Glu + ATP (right) are shown. (B) Bar graphs comparing the predicted and actual responses obtained at different holding potentials from coapplication of agonists for P2X2 and NMDARs, normalized to the sum of the separate Glu and ATP responses at each potential for each oocyte. The data are expressed as mean ± SEM; Statistical analysis performed using paired t-test * p < 0.05, *** p < 0.001. Parentheses denote number of replicates.
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Figure A5. P2X4–NMDAR inhibition is observed at low current responses. Representative current recorded from an individual oocyte expressing P2X4 and GluN2B-containing NMDARs, responding to: Glu (2 µM), ATP (5 µM), or Glu and ATP (2 µM and 5 µM, respectively). The predicted additive response (grey line) is calculated as the sum of the individual Glu and ATP induced currents.
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Figure A6. Altered response to Glu after P2X4–NMDAR coactivation. Representative current recorded from individual oocytes expressing P2X4 and NMDARs, responding to 2 µM Glu before (left) and after (right) Glu + ATP coactivation, in the presence (a) or absence (b) of calcium.
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Figure 1. P2X4–NMDAR coactivation produces an inhibited response. (a) Representative currents recorded from an individual oocyte coexpressing P2X4 and GluN2B-containing NMDARs responding to: Glutamate (Glu, 2 µM), ATP (5 µM), or Glu and ATP (2 µM and 5 µM, respectively). The predicted additive response (grey line) is calculated as the sum of the separate Glu and ATP induced currents. (b) Bar graphs comparing the predicted and actual responses obtained from coapplication of agonists for P2X4 and NMDARs containing GluN2A (n = 9), GluN2B (n = 9), or GluN2C (n = 10), normalized to the sum of the separate Glu and ATP responses for each oocyte. The data are expressed as mean ± SEM; Statistical analysis performed using paired t-test ** p < 0.01, **** p < 0.0001. P2X: Purinergic P2X receptors; NMDAR: N-methyl-d-aspartate receptor.
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Figure 2. P2X2–NMDAR coactivation produces an inhibited response. (a) Representative current recorded from an individual oocyte coexpressing GluN2B-containing NMDARs and P2X2 responding to 100 µM: Glu (left), ATP (middle), or Glu + ATP (right) are shown. (b) Bar graphs comparing the predicted and actual responses obtained from coapplication of agonists for P2X2 and NMDARs containing GluN2A (n = 22), GluN2B (n = 21), or GluN2C (n = 6), normalized to the sum of the separate Glu and ATP responses for each oocyte. (c) Representative current from an individual oocyte coexpressing P2X2 and NMDARs containing GluN2B. For sequential activation of P2X2 and NMDARs, primary application of either ATP (left) or Glu (right) first appears to reduce subsequent coactivation responses. The predicted response when ATP is applied first is calculated as the sum of the current response to ATP immediately before Glu is coapplied and the maximum current response to Glu thereafter. This order is reversed when calculating the predicted response when Glu is applied first. (d) Bar graphs of P2X2 and GluN2A (n = 16) or GluN2B (n = 17) containing NMDARs, comparing the predicted and actual responses obtained from sequential activation and coapplication of agonists, normalized to the sum of the predicted current. The data are expressed as mean ± SEM; Statistical analysis performed using paired t-test * p < 0.05, **** p < 0.0001.
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Figure 3. P2X4–NMDAR cross-talk is independent of calcium. (a) Representative currents recorded in Calcium-free Ringers’ solution (CfRS) from an individual oocyte coexpressing P2X4 and GluN2B-containing NMDARs responding to: Glu (2 µM), ATP (5 µM), or Glu and ATP (2 µM and 5 µM, respectively). The predicted additive response (grey line) is calculated as the sum of the individual Glu and ATP induced currents. (b) Bar graphs representing the predicted and actual responses obtained from coapplication of agonists, normalized to the sum of the separate Glu and ATP responses for each oocyte. For GluN2A, coactivation produced a statistically lower response than the predicted response (p < 0.01; paired t-test; n = 10). The same result was observed for GluN2B (p < 0.001; paired t-test; n = 9) and GluN2C (p < 0.0001; paired t-test; n = 28) coactivation. Furthermore, the degree of inhibition was not significantly different between the different GluN2 subunits (one-way ANOVA; p > 0.05) The data are expressed as mean ± SEM; statistical analysis performed using paired t-test, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Figure 4. The time-course for recovery of P2X4-mediated NMDAR inhibition in the absence or presence of calcium. (a) Representative current evoked by application of Glu (2 µM), before and after activation of P2X4 by ATP (5 µM), from an individual oocyte coexpressing GluN2B-containing NMDARs and P2X4 in the absence of calcium (CfRS). Bar graphs representing the mean of the amplitude of NMDARs responses to Glu before and after P2X4 activation by ATP, either in the absence (b) or presence (c) of calcium. All values were normalized to the Glu response obtained before P2X4 stimulation. Glu responses after P2X4 activation were significantly lower NMDARs containing each of the GluN2A-C subunits in the absence of calcium (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Tukey’s post-hoc test; n = 10–17 oocytes). Additionally, the time course of glutamate current recovery appears distinct, i.e., GluN2-subunit specific. The data are expressed as mean ± SEM.
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Figure 5. P2X c-terminus (CT) mediate interactions with NMDARs. (a) Top: A representative illustration of a homotrimeric P2X. The insert illustrates the differences in the size of the P2X4 (black) and P2X2 (grey) CT. (b) Bar graphs representing the predicted and actual responses obtained from coapplication of Glu (2 µM) and ATP (5 µM) in oocytes expressing either the P2X2 CT or the P2X4 CT, in combination with P2X2s or P2X4s and NMDARs. Agonist responses were normalized to the sum of the individual Glu and ATP responses for each oocyte. There was no statistically significant difference between the predicted responses and the actual responses produced by GluN2A-containing NMDARs and P2X4s in the presence of the P2X2 CT (105.0 ± 5.5%, p > 0.05, n = 7) Similarly, there was no statistically significant difference between the predicted responses and the actual responses produced GluN2A-containing NMDARs and P2X2s in the presence of the P2X4 CT (96.2 ± 3.9%, p > 0.05, n = 10) or the P2X2 CT (106.0 ± 6.21%, p > 0.05, n = 2). The data are expressed as mean ± SEM. Statistical analysis performed using paired t-test.
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Figure 6. Residues in the P2X4 CT confer the ability to interact with NMDARs. (a) An illustration of the mutations performed on the P2X4 internalization motif, compared to the wildtype P2X4 (P2X4WT); (b) Representative currents recorded in Ringers’ solution from an individual oocyte coexpressing P2X4-377TR and GluN2A-containing NMDARs responding to: Glu (2 µM), ATP (1 µM), or Glu and ATP (2 µM and 1 µM, respectively). The predicted additive response (grey line) is calculated as the sum of the individual Glu and ATP induced currents. (c) Bar graphs representing the current inhibition obtained from coapplication of Glu and ATP for oocytes coexpressing different P2X4 mutants and NMDARs. Agonist responses were normalized to the sum of the individual Glu and ATP responses for each oocyte and subtracted from 100%. The data are expressed as mean ± SEM. P2X4 CT mutations were statistically significantly different (p < 0.05) from the previously obtained inhibited coactivation responses for each GluN2 subunit (Kruskal–Wallis test with Dunn’s post-hoc analysis). ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Parentheses denote number of replicates.
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Figure 7. 11C peptide disrupts P2X–NMDAR cross-talk. Bar graphs representing the current inhibition obtained from coapplication of Glu and ATP for oocytes expressing either P2X4 (a) or P2X2 (b) and NMDARs containing GluN2A-C, 30 min after injection with 11C (grey). The inhibited responses (black) are ablated by 11C. Agonist responses were normalized to the sum of the individual Glu and ATP responses for each oocyte and subtracted from 100%. The data are expressed as mean ± SEM and were analyzed using a Welch’s t-test. ns p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Parentheses denote number of replicates.
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